SUMMARY Most respiratory diseases are thought to result from an aberrant or a lack of efficient repair mechanisms following injuries. Identifying the cellular sources and the mechanisms that can enhance the endogenous regeneration and that can aid cell-based therapies is much needed. In our recent published work, we identified a novel reserve multipotent stem cell population in the airway tissues. We found that myoepithelial cells that reside in the submucosal glands (SMG) of the airways are normally very quiescent but they proliferate extensively and migrate to repopulate surface airway epithelium (SAE) following severe damage. We further identified SOX9-mediated transcriptional programs are necessary for the proliferation and migration of SMG-derived myoepithelial cells to SAE. Furthermore, we found that a fraction of SMG-derived cells have the ability to fully convert into SAE, albeit very slowly. Our current preliminary data indicated that the fraction of SMG-derived cells that do not convert into SAE cells continue to maintain SMG cell characteristics, including the expression of transcription factor SOX9, for extended time periods. In addition, using newly developed mouse models and intra-vital imaging studies our preliminary data indicate that SMG-acinar luminal cells (serous and mucous cells) also have the ability to migrate to SAE. Based on our preliminary data, we hypothesize that SMG-acinar luminal cells have the ability to migrate and contribute to SAE regeneration and that this process is dependent on MECs migration. We also hypothesize that SOX9-dependent mechanisms must be downregulated for the complete conversion of SMG-derived cells into SAE cells. The major objectives of this proposal are to address both the potential contribution of SMG- acinar luminal cells to surface epithelium and to enhance proper regeneration. In Aim1, we will qualitatively and quantitatively determine the contribution of SMG-acinar luminal cells to SAE repair after injury. We will use our newly developed in vivo lineage tracing mouse models coupled with multi-photon assisted intravital imaging to determine the ability of SMG-acinar luminal cells migration and contribution to SAE repair. We will also use diphtheria toxin mediated ablation of MECs to test our hypothesis that SMG-acinar luminal cell proliferation and migration is dependent on MECs. In Aim2, we will test our hypothesis that loss of SOX9 is sufficient for the complete conversion of SMG-derived cells into SAE cells in the context of acute injury and chronic inflammation. This work has taken on added importance, as we found that SMG-derived cells contribution to SAE regeneration occurs in multiple forms of injury contexts including, influenza virus, Sulphur dioxide, chlorine, and chronic allergen- induced airway damage. Therefore, the outcomes from the proposed studies will have broader significance to airway diseases that occur due to defective regeneration. This work will lay the foundation for future studies involving human airway regeneration.